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Transparent brains and a revolution of studying every organ and tissue

Stanford psychiatrist Karl Deisseroth wants to know might be going wrong inside the heads of his patients. To study related conditions in animals, he previously developed the technique known today as optogenetics, which uses light to stimulate, and ideally to record from, the brain. The problem is fatty tissue like membranes and myelin, scatter the light. Other components, like heme in the blood of living tissue also absorb light. When appropriately stimulated, some proteins can actually give off a bit of light of their own in the form of fluorescence. To solve a few of these problems, researchers have sought ways to make the brain transparent. Yesterday, Deisseroth and his lab published the most detailed images of whole mouse brains that we have seen to date. To understand how this was achieved, we need look no further than a few techniques that have already been used for decades in the labs of neuroscientists and biochemists.

The traditional way of looking at cells is to slice the tissue into thin sections so they can be imaged with a microscope. In order to firm up the soft brain tissue, formaldehye is introduced into the brain to cross-link the proteins and nucleic acids in the tissue into a strong matrix. The method normally employed to get the formaldehyde into all the nooks and crannies is to use the circulatory system in a process called perfusion. A cut is made in the right atrium of the heart for the blood to drain out, and the formaldehyde is pumped in through the left ventricle.

Often in labs right next door to those of the neuroscientists busy perfusing brain tissues, biochemists and geneticists were busy fixing up solutions of acrylamide to make hydrogels to analyze DNA, RNA, and proteins. Using a process called electrophoresis, a voltage applied across a gel loaded with these components would pull them down the gel and separate them neatly according to size. This technique was how the DNA for the Human Genome Project was initially sequenced. The insight of Deisseroth, was that you could actually electrophorese the whole brain. In other words, fix everything you want with the formaldehyde, stabilize it in a hydrogel by reacting with acrylamide, and then pull all the light-blocking lipid out with electrophoresis. The only other important major steps left out were to break up these lipids with detergents so they don’t pull out other contents along with them.

Deisseroth has named his technique, Clarity, for Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel. Other researchers have tried to clarify brains in the past by lipid extraction techniques, but they usually ended up removing almost half the protein as well. The Clarity technique removed only around 8% of it.

The Clarity technique is not a direct component of the new BRAIN Initiative, although it will radically transform neuroscience. Whole swaths of departments will lose funding and be made obsolete, while others re-purposed. The study of not just the brain, but of every organ and tissue of the body, will be revolutionized. Pathologists will be able to see whole tissue, and the vast tissue banks of already preserved brains also can be clarified. Acquiring a connectome will now be much easier. In fact, Deisseroth has already clarified parts of the preserved brain of an autistic boy and is working on the rest to better understand the etiology of the disease. Next on the list might as well be the preserved brain of Einstein himself. Naturally, there are huge incentives to commercialize the technology, and patents have already been filed. Apparently, researchers and not-for-profit groups will have open access to the techniques though, and indeed they are right there in the published paper inNature.

The most powerful aspect of the technique is that it is compatible with existing methods of visualizing the components of neurons. To get the images we see, the mouse brains were labelled with different versions of fluorescent proteins, which attach selectively to different kinds of cells in the brain. Unlike the recent imaging studies we have just seen for the normally transparent zebrafish larva watching a paramecium, there is no activity in the fixed mouse brains. As a research animal then, it seems they will live another day since they are transparent in the living state. The zebrafish does beg the important next question, which perhaps should be the new central preoccupation in neuroscience, how do we make a large living brain transparent?

As any neuroscientist will tell you, that is impossible — at least for the moment. Others have suggested that perhaps we could just eat a lot of fast food (french fries contain some acrylamide), and take lots of statins (cholesterol-reducing drugs that are apparently a little bit indiscriminate in the lipid components they combat — also known as “muscle soreness”). Kidding aside, looking at these new images and videos of the brain, any non-jaded neuronaut must see in them the whole of mankind, and its future, laid bare. The task ahead is to make a mammalian brain into a zebrafish. A first step, a least in a temporary capacity, would be to use adequate substitute for the light absorbing heme in the blood. The icefish apparently has done this long ago, and survives by pumping its blood several times faster than normal in an environment where extreme pressure can help force oxygen into the blood. For us, it is more likely that artificial respirocytes, perhaps bearing small amounts of oxygen under pressure, might be used.

Replacing membranes and vesicles of living cells is just not going to happen. These structures are the structures of life. Replacing myelin, though, is perhaps a little less unthinkable. Before mammals arrived on the scene, most creatures got by with little or no myelated structure. While still metabolically active, myelin — once formed — appears to be largely static, even crystalline. It is a huge oversight that more funding focus is not placed on understanding anything at all about it. Student neuroscientists are simply told that myelin “increases membrane capacitance” and the whole possible science of the tissue is thereby dismissed. The truth is far beyond that child-like idea.

The Clarity technique is much more than a method of making brains invisible. The real technology here is as much the imaging and visualization power, as it is the chemical techniques. The resolution of the imaging is currently limited by the working distance of the microscope objective lenses used. Thinking out of the box with regard to lensing, and using other computational techniques like compressed sensing, will probably need to be used eventually. Solving these issues will give us images of subcellular details. For now, the technique is compatible with standard electron microscopy techniques, and imaged animals brains can later be co-registered with EM images to give a more complete brain data set. These efforts will probably occupy the immediate focus for some time. One thing to keep in mind is that if we can build a transparent cell phone, how hard can it be to do the same for a brain? After that, things will undoubtedly start start to get very interesting.